Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13-4 gene have recently been described in patients with FHL. We sequenced the Munc13-4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13-4 mutations were found, spread throughout the gene. Among novel mutations, 2650C-->T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13-4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

The immunological synapse of CTL contains a secretory domain and membrane bridges.

Cytotoxic T lymphocytes (CTL) rapidly destroy their targets. Here we show that although target cell death occurs within 5 min of CTL-target cell contact, an immunological synapse similar to that seen in CD4 cells rapidly forms in CTL, with a ring of adhesion proteins surrounding an inner signaling molecule domain. Lytic granule secretion occurs in a separate domain within the adhesion ring, maintaining signaling protein organization during exocytosis. Live and fixed cell studies show target cell plasma membrane markers are transferred to the CTL as the cells separate. Electron microscopy reveals continuities forming membrane bridges between the CTL and target cell membranes, suggesting a possible mechanism for this transfer.

Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail.

Fas ligand (FasL) induces apoptosis through its cell surface receptor Fas. T lymphocytes and natural killer cells sort newly synthesised FasL to secretory lysosomes but, in cell types with conventional lysosomes, FasL appears directly on the plasma membrane. Here, we define a proline-rich domain (PRD) in the cytoplasmic tail of FasL that is responsible for sorting FasL to secretory lysosomes. Deletion of this PRD results in cell surface expression of FasL in cells with secretory lysosomes. Positively charged residues flanking the PRD are crucial to the sorting motif and changing the charge of these residues causes mis-sorting to the plasma membrane. In cells with conventional lysosomes, this motif is not recognised and FasL is expressed at the plasma membrane. The FasL PRD is not required for endocytosis in any cell type, as deletion mutants lacking this motif are endocytosed efficiently to the lysosomal compartment. Endogenous FasL cannot internalise extracellular antibody, demonstrating that FasL does not transit the plasma membrane en route to the secretory lysosomes. We propose that an interaction of the PRD of FasL with an SH3-domain-containing protein, enables direct sorting of FasL from the Golgi to secretory lysosomes.

Normal and abnormal secretion by haemopoietic cells.

The secretory lysosomes found in haemopoietic cells provide a very efficient mechanism for delivering the effector proteins of many immune cells in response to antigen recognition. Although secretion shows some similarities to the secretion of specialized granules in other secretory cell types, some aspects of secretory lysosome release appear to be unique to melanocytes and cells of the haemopoietic lineage. Mast cells and platelets have provided excellent models for studying secretion, but recent advances in characterizing the immunological synapse allow a very fine dissection of the secretory process in T lymphocytes. These studies show that secretory lysosomes are secreted from the centre of the talin ring at the synapse. Proper secretion requires a series of Rab and cytoskeletal elements which play critical roles in the specialized secretion of lysosomes in haemopoietic cells.

Rab27a is required for regulated secretion in cytotoxic T lymphocytes.

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

Fratricide among CD8(+) T lymphocytes naturally infected with human T cell lymphotropic virus type I.

Infection and gene expression by the human T lymphotropic virus type I (HTLV-I) in vivo have been thought to be confined to CD4(+) T lymphocytes. We show here that, in natural HTLV-I infection, a significant proportion of CD8(+) T lymphocytes are infected by HTLV-I. Interestingly, HTLV-I-specific but not Epstein-Barr virus-specific CD8(+) T lymphocytes were shown to be infected. Furthermore, HTLV-I protein expression in naturally infected CD8(+) T lymphocytes renders them susceptible to fratricide mediated by autologous HTLV-I-specific CD8(+) T lymphocytes. Fratricide among virus-specific CTLs could impair the immune control of HTLV-I and possibly other lymphotropic viruses.

Sorting out the multiple roles of Fas ligand.

Fas ligand can both be used by the immune system to initiate cell death, and be used by non-lymphoid cells to evade death. Recent work has shown that Fas ligand is differentially sorted in different cell types. Here we present the viewpoint that the differential sorting plays an important part in determining the role of Fas ligand in different cells.

Analysis of the lysosomal storage disease Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder of human, mouse (beige) and other mammalian species. The same genetic defect was found to result in the disease in all species identified, permitting a positional cloning approach using the mouse model beige to identify the responsible gene. The CHS gene was cloned and mutations identified in affected species. This review discusses the clinical features of CHS contrasting features seen in similar syndromes. The possible functions of the protein encoded by the CHS/beige gene are discussed, along with the alterations in cellular physiology seen in mutant cells.

Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients.

The lytic proteins mediating target cell killing are stored in the lysosomes of activated cytotoxic T lymphocytes (CTL) and are secreted upon recognition of a target cell. These secretory lysosomes cannot be detected in resting T lymphocytes. Interaction of a resting cell with a target cell activates de novo formation of secretory lysosomes. CTL clones in culture mimic this behaviour, and so provide an ideal system for studying secretory lysosome biogenesis and maturation. In the genetic disease, Chediak Higashi syndrome (CHS), all lysosomes in the cells are enlarged and reduced in number compared with wild-type (WT) cells. We have used CTL from this disease to study secretory lysosome biogenesis and maturation. We show that at early stages after activation the secretory lysosomes are identical in WT and mutant cells, and that delivery of proteins to the secretory lysosome along the biosynthetic and endocytic pathways is normal in the mutant cells. With time, the lysosomes in the mutant cells aggregate, become larger and fewer in number and eventually form giant structures. Our results show that the initial steps of secretory lysosome formation are normal in CHS, but that the organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes.

Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells.

Fas ligand (FasL) triggers apoptosis during cytotoxicity mediated by cytotoxic T lymphocytes and during immune downregulation. The ability of T cells and natural killer cells to trigger apoptosis through this mechanism is controlled by the cell surface expression of FasL (ref. 2). Because FasL expression is up-regulated on activation, FasL was thought to be delivered directly to the cell surface. Here we show that newly synthesized FasL is stored in specialized secretory lysosomes in both CD4+ and CD8+ T cells and natural killer cells, and that polarized degranulation controls the delivery of FasL to the cell surface. In this way, FasL-mediated apoptosis is finely controlled by receptor-mediated target-cell recognition. The cytoplasmic tail of FasL contains signals that sort FasL to secretory lysosomes in hemopoietic cells. This pathway may provide a general mechanism for controlling the cell surface appearance of proteins involved in immune regulation.

Enhanced interaction of HLA-DM with HLA-DR in enlarged vacuoles of hereditary and infectious lysosomal diseases.

Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.

L is for lytic granules: lysosomes that kill.

CTL are important cells in the immune system which are able to recognise and directly destroy virally infected, tumorigenic or foreign cells. The proteins which mediate this destruction are packaged into specialised secretory granules, termed lytic granules, which are secreted in response to target cell recognition. Curiously these specialised secretory granules also contain all the lysosomal hydrolases, and in CTL the lytic granules serve two separate functions: as a lysosome within the cell, and as a secretory granule when a target cell is recognised. These "secretory lysosomes", which serve important roles in both protein degradation within the cells as well as regulated secretion of proteins from the cells, are also found in other cell types, all of which are derived from the hemopoietic lineage. This observation raises the possibility that cells of the hemopoietic lineage possess specialised sorting and secretory mechanisms which allow the lysosomes to be used as secretory organelles. Studies on Chediak Higashi syndrome support this idea, since in this naturally occurring genetic mutation, cells with secretory lysosomes are unable to secrete their granules while other conventional secretory cells are able to do so. Further studies on the mechanisms which regulate secretion of lytic proteins from CTL should identify the proteins involved in this unusual secretory pathway. Some aspects of the differences between conventional and "secretory" lysosomes remain unresolved. How the biogenesis of the secretory lysosome differs from that of a conventional secretory granule is unclear. While conventional secretory cells sort proteins destined for the granule by a selective condensation in the TGN, the secretory lysosomes seem to use a combination of lysosomal and other sorting signals. Our preliminary studies suggest that haemopoietic cells possess specialised sorting mechanisms which allow the correct sorting of the secreted products to the lysosome, and that these signals are different from those found in conventional secretory (e.g. neurosecretory) cells. This finding and the observation that fibroblast lysosomes can undergo calcium-mediated exocytosis suggests that the unusual secretory system found in haemopoietic cells may be a result of specialised sorting mechanisms in these cells. In this case the Chediak lesion may turn out to be a sorting defect.

Activated CD4+ and CD8+ cytotoxic cells are present in increased numbers in the intestinal mucosa from patients with active inflammatory bowel disease.

The contribution of cell-mediated cytotoxicity to the pathogenesis of inflammatory bowel disease (IBD) is controversial, and results of in vitro assays vary according to experimental procedures. Therefore, we compared the frequency of cytotoxic effector cells in situ. On tissue sections of controls (n = 11), low frequencies of granzyme A and perforin mRNA-expressing cells are found in the lamina propria (1.77 +/- 0.15% and 1.46 +/- 0.12%, respectively) and in the epithelial cell layer (0.76 +/- 0.12% and 0.66 +/- 0.10%, respectively). In patients with IBD (n = 33), corresponding values were significantly (P < 0.02) higher, 6.1 +/- 0.40% and 5.92 +/- 0.57% for granzyme A and perforin expression in the lamina propria and 2.50 +/- 0.19% and 2.59 +/- 0.28%, respectively, in the epithelial compartment. Differences between ulcerative colitis and Crohn's disease are statistically not significant (P > 0.33). Activated cytotoxic cells are preferentially found at sites facing the intestinal lumen. Perforin mRNA-expressing cells are mainly CD8+ T cells. CD4+ T cells expressing perforin mRNA are mainly isolated from affected areas of patients with Crohn's disease. Immunostaining for perforin protein generally coincides with perforin mRNA in situ. These data demonstrate that cytotoxic cells are vigorously activated in situ in the intestinal mucosa of patients with active IBD.

Protein sorting and secretion during CTL killing.

Now that the key proteins involved in target cell destruction during the lethal hit from CTL have been identified, an understanding of the mechanisms which regulate their delivery becomes important for understanding their relative contributions during CTL killing. This review summarizes what is known about the packaging and delivery of the different effector proteins involved in target cell destruction. The mechanisms which regulate their delivery during killing contribute to some of the unusual features of CTL killing, and give some clues about the different contributions in target cell destruction.

Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain.

Perforin is a secreted protein synthesized by activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It is a key component of the lytic machinery of these cells, being able to insert into the plasma membrane of targeted cells, forming a pore which leads to their destruction. Here we analyse the synthesis, processing and intracellular transport of perforin in the NK cell line YT. Perforin is synthesized as a 70 kDa inactive precursor which is cleaved at the C-terminus to yield a 60 kDa active form. This proteolytic cleavage occurs in an acidic compartment and can be inhibited by incubation of the cells in ammonium chloride, concanamycin A, leupeptin and E-64. The increased lytic activity of the cleaved form can be demonstrated by killing assays in which cleavage of the pro-piece is inhibited. Epitope mapping reveals that cleavage of the pro-piece occurs at the boundary of a C2 domain, which we show is able to bind phospholipid membranes in a calcium-dependent manner. We propose that removal of the pro-piece, which contains a bulky glycan, allows the C2 domain to interact with phospholipid membranes and initiate perforin pore formation.

Secretory lysosomes - a special mechanism of regulated secretion in haemopoietic cells.

The secretory granules o f cells derived from the haemopoietic lineage are 'secretory lysosomes' containing both lysosomal hydrolases and secretory proteins. Studies on cytotoxic T lymphocytes (CTLs) have elucidated several of the mechanisms that regulate protein sorting to, and secretion from, this unusual secretory organelle. In particular, recent findings from a CTL mutant have led to the hypothesis that CTLs, and other cells of the haemopoietic lineage, use specialized sorting and secretory mechanisms in which the lysosome functions as a regulated secretory granule.

Loss of cytotoxic T lymphocyte function in Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis.

CTLs from patients with Chediak-Higashi syndrome (CHS) are unable to destroy target cells recognized via the TCR. To determine the mechanism responsible for the loss of cytotoxicity, CD8+ CTL clones have been derived from a patient with CHS. Individual CTL clones show poor killing that can be increased in longer assays. However, in the presence of cycloheximide, the small amount of killing observed is abolished, indicating killing arises from newly synthesized proteins, rather than from proteins stored in granules. In this study, we show that the CHS CTL clones express normal levels of the lytic proteins granzyme A, granzyme B, and perforin, which are processed properly during biosynthesis and targeted correctly to giant lytic granules. Despite the difference in size, CHS and normal lytic granules are similar, in that both contain the lysosomal enzyme cathepsin D and the lytic protein granzyme A, and lack the mannose-6-phosphate receptor (MPR). However, unlike normal CTL clones, the CHS CTL clones are unable to secrete their giant granules in which the lytic proteins are stored. After cross-linking the TCR, CHS CTL clones fail to secrete granzyme A, as assayed by both enzyme release and confocal microscopy. We suggest that the defect in CHS lies in a protein that is involved in membrane fusion and is essential for the secretion of lysosomal compartments in certain hemopoietic cells.

The cell biology of CTL killing.

A great deal is known about the immunology of cytotoxic T lymphocyte killing, but much less is known about the cell biology of this process. Recent work has begun to elucidate the mechanisms that control lytic-protein secretion, and reveals that some unusual features of these secretory processes might explain some of the important features of killing by cytotoxic T lymphocytes.